1. Field of the Invention
The present invention relates to a method of producing a novel secondary alcohol dehydrogenase useful for the preparation of alcohol, aldehyde and ketone, especially for that of an optically active alcohol, a method of producing said enzyme, a DNA segment encoding said enzyme, a microorganism transformed with said DNA, and a method of producing alcohol, aldehyde and ketone, especially optically active alcohol using said enzyme.
2. Related Arts
Of the secondary alcohol dehydrogenase of the microbial origin requiring nicotinamide adenine dinucleotide phosphate (abbreviated as NADP.sup.+ hereinafter), the one derived from Thermoanaerobium brockii is well documented (J. Am. Chem. Soc. 108, 162-169 (1986)). In addition, of the secondary alcohol dehydrogenase requiring nicotinamide adenine dinucleotide (abbreviated as NAD.sup.+ hereinafter), there have been reported those derived from Pichia sp. NRRL-Y-11328 (Eur. J. Biochem. 101, 401-406 (1979)), Pseudomonas sp. SPD6 (Bioorg. Chem. 19, 398-417 (1991)), Pseudomonas fluorescence NRRL B-1244 (Tokkai Sho, 59-17982), Pseudomonas maltophilia MB11L (FEMS Microbiol. Lett. 93, 49-56 (1992)), Pseudomonas sp. PED (J. Org. Chem. 57, 1526-1532 (1992)), Pseudomonas sp. ATCC 21439 (Eur. J. Biochem. 119, 359-364 (1981)), Candida boidinii SAHM (Biochim. Biophys. Acta 716, 298-307 (1992)), Mycobacterium vaccae JOB-5 (J. Gen. Microbiol. 131, 2901-2907 (1985)), Rhodococcus rhodochrous PNKb1 (Arch. Microbiol. 153, 163-168 (1990)), Comamonas terrigena (Biochim. Biophys. Acta 661, 74-86 (1981)), and Arthrobacter sp. SBA (Tokkai Sho 51-57882).
However, the stereochemical substrate specificity of these secondary alcohol dehydrogenases is not satisfactory for the practical application. For example, as to 2-butanol, one of the most frequently reported substrates of the secondary alcohol dehydrogenase, there has not been reported the enzyme which will oxidize (S)-2-butanol stereospecifically to produce 2-butanone. (The enzymes derived from Pseudomonas sp. ATCC 21439, Pseudomonas sp. SPD6, Comamonas terrigena, Candida boidinii SAHM or Pichia sp. NRRL-Y-11328 oxidize (R)-isomer preferentially, while the one derived from Pseudomonas fluorescens NRRL B-1244 does not show any definite substrate stereochemical specificity, and the specificity of the enzyme derived from Mycobacterium vaccae JOB-5, Rhodococcus rhodochrous PNKb1, Pseudomonas sp. PED or Pseudomonas maltophilia MB11L has not been reported.) Furthermore, although the primary alcohol dehydrogenase (SADH-1) derived from baker's yeast (Saccharomyces cerevisiae) has been reported to oxidize 2-butanol with S configuration preferentially, the relative activity is as low as about 1% of that for ethanol, not suitable for practical use (Arch. Biochem. Biophys. 126, 933-944 (1968), J. Biol. Chem. 268, 7792-7798 (1993)).
Since the secondary alcohol dehydrogenase which will preferentially oxidize S-2-butanol has not been reported, there has been a strong demand for finding the enzyme with a high substrate stereochemical specificity.
There has been also a high demand for cloning DNA encoding said enzyme, because it will be possible to produce said enzyme on a large scale with a genetic engineering technique using the cloned gene of said enzyme.